Biochemical and ultrastructural study of Leu M1 antigen in Reed-Sternberg cells: comparison with granulocytes and interdigitating reticulum cells.

A group of monoclonal antibodies was shown to react with glycoconjugates containing a sugar sequence--lacto-N-fucopentaose III (LNF-III)--in granulocytes and in some normal nonlymphoid cells. The antibodies including anti-Leu M1, anti-My-1, WGHS 29-1, 534F-8, and 538F-12 of the immunoglobulin M-type were used to study the biochemical properties of LNF-III antigens in granulocytes, interdigitating reticulum cells, and neoplastic cells of Hodgkin's disease. In contrast to the presence of an abundant LNF-III glycolipid in granulocytes, the Hodgkin's neoplastic cells had no LNF-III glycolipid or contained only minimal amounts; however, both LNF-III glycoconjugates isolated from Hodgkin's neoplastic cells and interdigitating reticulum cells appeared to be a similar, if not an identical, 150,000-molecular-weight glycoprotein. The neoplastic cells in Hodgkin's disease appeared to show a biochemical property more closely related to interdigitating reticulum cells than any other cells in the monocyte-granulocyte-histiocyte system.

Pattern of isoaccepting transfer RNAs common to 26 patients with Hodgkin's disease.

The elution profiles of aminoacyl transfer RNAs from Hodgkin's tumors have been compared with the corresponding patterns from normal splenic tissue. Aminohexyl-Sepharose and reversed-phase 5 chromatography have been used in the fractionation studies. Three peaks of acceptor activity have been observed in phenylalanyl, histidyl, aspartyl, and asparaginyl transfer RNAs. A second peak was shown in the case of tyrosyl and methionyl transfer RNAs. Seryl transfer RNA showed no change in elution profile; namely, a single species was observed in both normal and tumor transfer RNAs. These observations are confirmation that Hodgkin's disease is a malignant disease. The uniformity of the extra species of tRNA suggests that there is a commonly occurring aberration in the cell of origin of the Hodgkin's tumor.

Relative subunit composition of the ferritin synthesized by selected human lymphomyeloid cell populations.

Biosynthesis of ferritin subunits by cell sets isolated from normal human peripheral blood, spleens of Hodgkin's disease patients, and tumor cell lines were investigated. Normal mature hematopoietic cells made a ferritin with more H (21K) than L (19K) subunits. The reverse was found for a promyelocytic tumor cell line and tumor cell lines derived from other tissues. Two dimensional electrophoresis indicated H has a lower pI than L. Therefore relative proportions of the two subunits contribute to the electrophoretically distinct forms of the isoferritins. In response to increasing concentrations of iron in vitro, a selected monocyte population synthesized more H than L; L biosynthesis however increased more than H. Some possible regulatory implications of these observations are discussed.

Leu-M1 antigen in human neoplasms. An immunohistologic study of 400 cases.

Several studies have shown that the Leu-M1 antigen, a monocyte/granulocyte-related marker, is consistently expressed by the neoplastic cells of patients with Hodgkin's disease (HD). It has been suggested that reactivity of Reed-Sternberg cells with Leu-M1 can be used in support of a morphologic interpretation of HD, and that it is helpful in the differential diagnosis of HD from morphologically similar lesions. To evaluate the significance of the Leu-M1 positivity of Reed-Sternberg cells in the diagnosis of HD, we investigated the distribution of Leu-M1 antigen in a series of patients with HD, non-Hodgkin's lymphomas, and nonhematopoietic neoplasms. We were able to demonstrate the presence of Leu-M1 antigen not only in the majority of patients with HD, but also in 12 of 18 (67%) peripheral T-cell lymphomas, as well as in a variety of nonhematopoietic neoplasms, which included 113 of 199 carcinomas, most of them (58%) adenocarcinomas. Only one of 34 sarcomas showed a focal positive reaction. Leu-M1-related antigen was not detected in any of 18 mesotheliomas, 15 germ cell tumors, 13 melanomas, three schwannomas, or three astrocytomas. Our study indicates that Leu-M1 positivity has no value in supporting the diagnosis of HD in situations where the histologic diagnosis of HD is doubtful. However, since anti-Leu-M1 reacted positively in the majority of adenocarcinomas but was absent in mesotheliomas, melanomas, and most sarcomas, this antigen could serve as a new marker that may be helpful in situations in which carcinoma is a part of the differential diagnosis.

Demonstration of lactoferrin in tumor tissue from two patients with positive gallium scans.

We have detected lactoferrin in tumor tissue from a patient with Hodgkin's disease and a patient with Burkitt's lymphoma. Both patients had radiogallium scans demonstrating increased uptake in the tumor tissue subsequently found to contain lactoferrin. Tissue assay for lactoferrin was performed by the indirect immunofluorescence method. Control splenic tissue showed either slight lactoferrin content or none.

Immunoglobulin and T-cell receptor gene rearrangements in Hodgkin's disease and Ki-1-positive anaplastic large cell lymphoma: dissociation between phenotype and genotype.

We have determined the tumor cell immunophenotype and the rearrangement configuration of immunoglobulin and T-cell receptor genes in 39 cases of Hodgkin's disease (HD), six HD-derived cell lines and 22 cases of Ki-1-positive anaplastic large cell lymphomas (Ki-1-ALC). Rearrangements were observed in 11/39 HD cases, 15/22 Ki-1-ALC, and all cell lines. Epstein-Barr virus DNA was found in five HD cases, one cell line, and one Ki-1-ALC. Both HD and Ki-1-ALC frequently displayed a dissociated genotypic and phenotypic maturation status, i.e. an immature genotype in association with late activation markers. We postulate that the tumor cells in many cases of HD and some cases of Ki-1-ALC may be derived from immature lymphoid cells by a transformation process that superimposes characteristics of mature activated lymphocytes on these cells.

Monoclonal antibody Leu-22 (L60) permits the demonstration of some neoplastic T cells in routinely fixed and paraffin-embedded tissue sections.

Monoclonal antibody Leu-22 (L60) detects a T cell-associated antigen which is stably expressed in routinely fixed and paraffin-embedded tissue sections. We investigated the utility of monoclonal antibody Leu-22 to immunophenotype routinely processed lymphoid neoplasms by determining its reactivity in 105 archival pathologic specimens of lymphoid neoplasia that had been previously immunophenotyped by standard cell suspension and frozen tissue section techniques. Monoclonal antibody Leu-22 reacted with 69% of T cell non-Hodgkin's lymphomas (NHLs), including cases belonging to each of the major clinicopathologic categories, and with 22% of B cell NHLs, but did not react with the Reed-Sternberg (RS) cells of Hodgkin's disease (HD). We concluded that monoclonal antibody Leu-22 reacts preferentially but not exclusively with T cell NHLs. Therefore, we performed parallel analyses of the same 105 cases with monoclonal antibodies leukocyte common antigen (LCA), Leu-M1, LN1, and LN2, which detect various paraffin-resistant antigens, and of 80 of these cases with monoclonal antibody UCHL1, which detects a paraffin-resistant T cell-associated antigen. UCHL1 reacted with 61% of the T cell NHLs studied. Sixty-nine percent of T cell NHLs expressed the LCA+, Leu-22+ or Leu-M1+, LN1- phenotype and 47% of B cell NHLs expressed the LCA+, Leu-22-, Leu-M1-, LN1+ phenotype. These phenotypes had a false-positive rate of only 7%. The substitution of UCHL1 for Leu-22 or the combined use of UCHL1 and Leu-22 in this panel did not improve our ability to correctly predict the T cell phenotype of these lymphoid neoplasms. LN1 and LN2 reacted with 13% and 56% of T cell NHLs, respectively, and LN2 reacted with RS cells in 85% of cases of HD. In summary, our results demonstrate that the judicious use of monoclonal antibody Leu-22 in combination with other selected commercially available monoclonal antibodies permits the determination of the B cell or T cell origin of a high proportion of NHLs, and is helpful in the differential diagnosis between HD and NHL among cases that have been routinely fixed and paraffin-embedded.

Hodgkin's disease: a flow cytometric study.

Flow cytometry was performed on paraffin embedded tissue from 115 cases of Hodgkin's disease. Thirteen (11%) tumours were aneuploid with no significant difference between the histological subgroups. The median proliferative index was 14%, and the highest values were found in the NS2 (16.4%) and lymphocyte depleted (16.0%) subgroups. The difference in proliferative index approached significance when the NS2 subgroup was compared with the NS1 subgroup (p less than or equal to 0.11) and when the lymphocyte depleted and NS2 subgroups combined were compared with the mixed cellularity, lymphocyte predominance, and NS1 subgroups combined (p less than or equal to 0.07). There was a trend towards better survival for patients with aneuploid tumours and those cases with a proliferative index below 15%, but neither of these trends was significant.

Cells which contain S-100 protein in Hodgkin's disease: a quantitative study.

Paraffin sections from a series of 50 lymph nodes affected by Hodgkin's disease were examined by means of the unlabelled primary antibody peroxidase-antiperoxidase method to detect those cells which contained S-100 protein. In addition, 15 lymph nodes showing reactive follicular hyperplasia were studied. A simple enumeration procedure (eyepiece graticule) was used to count the number of such cells in 20 standard 25 X microscope fields. In the specimens of nodular sclerosing Hodgkin's disease many cells positive for S-100 protein were present, in contrast to the other Rye subtypes, which showed a relative paucity. By comparison, the lymph nodes showing reactive follicular hyperplasia contained a similar number of cells containing S-100 to those seen in the nodular sclerosing lymph nodes affected by Hodgkin's disease.

Transferrin receptors in human tissues: their distribution and possible clinical relevance.

The distribution of transferrin receptors (TR) has been studied in a range of normal and malignant tissues using four monoclonal antibodies, BK19.9, B3/25, T56/14 and T58/1. In normal tissues TR was found in a limited number of sites, notably basal epidermis, the endocrine pancreas, hepatocytes, Kupffer cells, testis and pituitary. This restricted pattern of distribution may be relevant to the characteristic pattern of iron deposition in primary haemachromatosis. In contrast to this limited pattern of expression in normal tissue, the receptor was widely distributed in carcinomas, sarcomas and in samples from cases of Hodgkin's disease. This malignancy-associated expression of the receptor may play a role in the anaemia of advanced malignancy by competing with the bone marrow for serum iron.

Ferritin H and L mRNAs in human neoplastic tissues.

The typical tissue isoferritin pattern varies during neoplastic transformation, usually shifting toward a more acidic profile. To investigate the molecular basis of this phenomenon, we have analyzed the steady-state levels of the H and L mRNAs in several neoplastic tissues. By using specific probes for the two ferritin subunits, we have found, in three different adenocarcinomas and in a case of Hodgkin lymphoma, a two- to four-fold increase of the H and L mRNA levels compared to those found in normal human liver.

Immunoprecipitation of the interleukin-2 receptor from Hodgkin's disease derived cell lines by monoclonal antibodies.

The nature of Hodgkin and Reed-Sternberg (H-RS) cells and their normal counterpart remains a matter of controversy. Our recent investigations have suggested that H-RS-cells derive from certain activated lymphocytes of either B or T cell origin. In keeping with this concept, we were able to demonstrate that in the majority of cases of Hodgkin's disease the interleukin-2 receptor (IL2-R) was detectable on H-RS-cells by three monoclonal antibodies (anti-Tac, Tü69 and ACT-1) with the sensitive alkaline phosphatase anti-alkaline phosphatase (APAAP) tissue staining procedure. In extension of these studies, we precipitated IL2-R antigen from two established permanent cell lines (L540 and L591) derived from patients with Hodgkin's disease. Although these cell lines are different in nature, in that the L540 line shows rearrangement for the T cell receptor beta chain gene and the L591 line displays rearrangement for immunoglobulin genes (gamma heavy and lambda light chain), the antigen precipitated by monoclonal antibodies against IL2-R exhibited a molecular weight of approximately 53 kd on the L591 line and 58 kd on the L540 line. Thus, these data strongly support the view that the labelling of H-RS-cells in tissue sections for IL2-R was not merely due to cross-reactivities of the two anti-IL2-R antibodies with a determinant of an unrelated structure but represents true IL2-R molecules.

[DNA content of lymphocytes and mononuclear tumor cells in Hodgkin's disease]. / DNS-Gehalt von Lymphocyten und einkernigen Tumorzellen beim Morbus Hodgkin.

Thirteen lymph node biopsy specimens with Hodgkin's disease (six cases of the nodular sclerosis type; six cases of the mixed cellularity type; one case of the lymphocytic depletion type) were studied with scanning cytophotometric measurements. The DNA content of small lymphocytes, large lymphocytes, atypical mononuclear cells and Hodgkin cells was measured. The sizes of the nuclei and mitotic indices were also measured. There were no lymphocytes with aneuploid DNA stemlines. Large lymphocytes showed proliferative activity which is possibly reactive, although its neoplastic character cannot be excluded. Hodgkin cells exhibited aneuploid values in seven cases and atypical mononuclear cells in only four cases. The nuclear sizes and the DNA content of the atypical mononuclear cells ranged between the values of lymphocytes and Hodgkin cells. The mixed cellularity type showed higher DNA values, more frequently occurrence of aneuploid stemlines and a higher frequency of mitotic figures than did the nodular sclerosis type. The results confirm the different grades of malignancy of the various histopathological categories of Hodgkin's disease.

The distribution of iron and iron binding proteins in spleen with reference to Hodgkin's disease.

The distribution of iron and iron binding proteins (IBP) have been compared with control spleen tissue in an attempt to establish a pattern of staining restricted to Hodgkin's disease (HD). All but one of the HD spleens examined stained for ferritin, which was largely present in red pulp dendritic macrophages (DM). In spleens histologically involved with HD heavy deposits of ferritin were seen around tumour nodules. Staining for ferritin increased with involvement of the spleen in HD but DM still represented the bulk of positive cells. However, ferritin positive DM were frequently seen in control spleens, and often in large numbers. Staining of ferric iron by Perls technique was less prominent than ferritin but this observation was also true of the non-HD spleens studied. Patterns of staining with transferrin were equivalent in both groups of spleens with DM being the most frequently positive cell type. Polymorphous macrophages showing erythrophagocytosis were present in the red pulp sinuses of all groups of spleens and although these cells have been considered as precursors of the Reed-Sternberg cell their presence seemed related to total splenic ferritin regardless of the disease process. These cells marked as macrophages and their presence was not restricted to HD. The results show that there is no particular appearance of iron or IBP distribution which is restricted to HD spleens. However, staining for ferritin and iron increased in HD spleens with tumour involvement and could contribute to circulatory abnormalities in this disease.

Identification of peanut agglutinin binding glycoproteins restricted to Hodgkin's disease-derived cell lines.

Peanut agglutinin (PNA) binding glycoproteins from four Hodgkin's disease (HD)-derived cell lines and a variety of cell lines/peripheral blood cells representative of the lymphoid and myeloid lineages were identified by probing nitrocellulose membranes of SDS-PAGE separated NP40 solubilized cellular glycoproteins with [125I]-labelled PNA. The two Hodgkin's cell lines Ho and L428 demonstrated the most heterogeneous glycoprotein profiles each expressing 15 PNA binding glycoproteins, respectively. The two remaining Hodgkin's lines Co and L591 expressed only four glycoproteins each and these were all also commonly expressed by Ho and L428. Comparative analysis with all other cell types studied revealed the expression of five glycoproteins restricted to Ho (gp42, gp40, gp38, gp24 and gp22) and six restricted to L428 (gp180, gp75, gp40, gp38, gp24 and gp22). Four of these, gp40, gp38, gp24 and gp22 were commonly expressed by both Ho and L428. Of cell lines of myeloid lineage studied only the erythroleukemia cell line K562 expressed detectable glycoproteins also expressed by some of the Hodgkin's cell lines (gp110, gp96, gp50 and gp45). Only one glycoprotein, gp20 expressed by Ho was also commonly expressed by normal peripheral blood granulocytes. This limited study has thus succeeded in demonstrating for the range of cell types studied, that some glycoproteins with terminal D-galactose beta (1----3) N-acetyl galactosamine oligosaccharide sequences are apparently restricted to two of the HD cell lines. Moreover, the heterogeneous glycoprotein profiles obtained for the HD cell lines Ho and L428 suggests that galactosylation processes in these two cell lines is aberrant.

[Beta 2 microglobulin in some hematologic neoplasms]. / Beta 2 microglobulina en algunas neoplasias hematológicas.

Beta 2 microglobulin is a low molecular weight protein integrating the light chain HLA antigens. Its serum concentration is increased in different neoplasias and in renal failure. Using solid phase RIA we determined the concentration of beta 2 microglobulin in plasma and spinal fluid of 57 healthy individuals and patients with hematologic neoplasia. Serum levels were 1.34 +/- 0.34 mg/l and spinal fluid levels were 1.3 +/- 0.7 mg/l in healthy subjects. Serum levels in 29 patients with myeloma was 7.51 mg/l, significantly higher in those with renal failure (12.35 mg/l) compared to those without (4.54). In 30 patients with non-Hodgkin lymphoma the mean serum levels were 2.90 mg/l, significantly greater in those with active disease (3.18) than in those with remission (1.5). No difference was found according to the degree of malignancy. Patients with acute lymphatic leukemia had elevated values of beta 2 microglobulin while the disease was active (3.37 mg/l), decreasing to normal levels after remission (1.79 mg/l). Spinal fluid levels of beta 2 microglobulin were elevated only in patients with central nervous system involvement. Our results indicate that serum levels of beta 2 microglobulin are helpful in patients with hematologic neoplasia in assessing the activity of the disease and tumor mass, especially in multiple myeloma.

The distribution of fibronectin in lymph nodes infiltrated by Hodgkin's disease. An immunoperoxidase study on paraffin sections.

The tissue distribution of fibronectin (FN) was examined using a commercial anti-FN serum, the peroxidase-anti-peroxidase (PaP) technique, and paraffin sections of 22 lymph nodes affected by Hodgkin's disease. Vascular basement membranes and reticulin fibres are selectively stained and their structural changes in this pathological condition become readily visible. In contrast to the normal lymph node and to Hodgkin's disease with lymphocytic predominance, cases of mixed cellularity disease contain individual and focally grouped cells displaying intracytoplasmic FN. In nodular sclerosis these cells with fibroblast morphology are consistently numerous in the marginal zones of the cellular nodes. Strongly reacting mastocytes probably absorbed the applied antiserum non-immunologically. All the other cell types giving rise to the varying appearances of Hodgkin's lesions are consistently negative with respect to intracellular FN, including all forms of Hodgkin cells. We conclude that in Hodgkin's disease the immigration of FN-secreting fibroblasts is an integral part of the early sclerosing reaction, which in itself is a defence/repair mechanism closely related to scar formation.